Immune Status Study of Dentistry Student and Children
Naturally Infected with Measles Virus for Detection of Primary
and Secondary Immune Failures
Younis Abdulredha K. Al-Khafaji
Branch of Medical Microbiology, College of Dentistry, University of Babylon,
Iraq.
MJ B
Abstract
This study was carried out on 52 subject of Babylon dentistry student and children who were naturally
infected with measles virus and laboratory confirmed as measles during 2009 by detection of specific
anti-measles IgM .They were tested for IgG avidity to estimate their immune status and to distinguish
between primary measles infection and reinfection due to secondary vaccine failure. The study
reflected that all unvaccinated subjects (19.2%) showed a primary immune response whereas
secondary immune responses were detected in( 46.09%). Of 34 subjects sourly vaccinated (42.9 %) of
them reflected a primary immune response, whereas (57.1%) of them reflected secondary immune
response ,thereby indicating a secondary vaccine failure that was seen at very high proportion in
subjects > 11 years old, so that revaccination of 11 years old children is recommended for reactivation
of their immune status to restrict secondary reinfection.
‫دراسة الحالة المناعية لطلبة كلية طب األسنان وألطفال المصابين طبيعيا بفيروس الحصبة‬
.‫لتحديد فشل االستجابة المناعية األولية والثانوية‬
‫الخالصة‬
‫وقبد‬2009 ‫تم دراسة الحالة المناعية لطلبة كلية طب األسنان واألطفال المصابين طبيعيا بفيروس الحصببة يبب باببال بلل العبام الد ارسبب‬
‫ كمببا تببم التحبري عببن النلوييببولين‬M ‫ إصبابة تببم تص يصببتا م تبريبا مببن ببلل التحبري عببن النلوييببولين المنباعب‬52 ‫الد ارسببة علب‬
‫الد ارسبة ببان‬
‫أجريب‬
‫ بنية التفريق بين اإلصابة األولية بفيروس الحصبة ويين عودة اإلصابة بسببب الفصبال النبانوي للل باو ك وعكسب‬G ‫المناعب‬
‫)مبنتم اسبتجابة مناعيبة نانويبة كةصبارة إلب‬%57.1( ‫) أعطوا استجابة مناعية أوليبة بينمبا أببد‬%19.2( ‫جميع المرض غير المطعمين‬
‫يصال نانوي للل او والذي لوحظ بنسبة عالية جدا يب األعمار أكنبر مبن احبد عصبر عامبا وعليبأ أر ضبرورة إعبادة تطعبيم األطفبال بتبذا‬
‫العمر لتنصيط الحالة المناعة لديتم للحد من العدو النانوية بالفيروسك‬
‫بببببببببببببببببببببببببببببببب‬
Introduction
easles is a highly communicable
infections disease , it remains
the leading cause of vaccine
preventable childhood mortality in
developing countries, and is still a
major public health concern in the
developed countries [14]
Measles virus (MV) is a
member
of
the
family
paramyxoviridae, genus Morbillivirus.
It is transmitting via the respiratory
M
rout and has an incubation phase of 9
to 19 days [5].
Measles outbreaks are known
to occur even in highly vaccinated
population despite the availability of
an effective live attenuated measles
virus vaccine [19]. Mild or
asymptomatic measles infection are
probably very common among
measles- immune persons exposed to
measles cases and may be the most
common manifestation of measles
during outbreaks in highly immune
populations [9] .
Measles control has a high priority in
many countries, and it is important that
questions surrounding possible vaccine
failures eliminate measles may be
evaluated and strengthened [20].
A study from the united states
of 80 blood donors of whom 8
developed measles during an outbreak
in a university college , reported that
individual with low measles antibody
titers ( ≤ 120 m IU) were susceptible
to infection [2] .
Observation from immunized
populations suggest that undetectable
antibodies may not necessarily imply
that the individual is fully susceptible
to disease [1] ,[13].
Measles virus-specific high-avidity
antibodies are associated with preexisting B-cell memory, whereas low
avidity IgG is an indication of the
primary immune response [18], [7] ;
[21] .Thus avidity measurement can be
used to assess the success of measles
vaccination [3],[7] and offers a way of
assessing the type of vaccine failure
without knowledge of prior antibody
status [16]. In this study, we used IgG
avidity assay to analyze sera from
conformed measles patient in order to
determine how many cases of
reinfection (high- avidity antibodies)
and how many were cases of primary
infection (low- avidity antibodies) in
adult subject and study of there
immune response.
It is important that highly
sensitive andspecific laboratory test
Enzyme Immune Assay (EIA) used to
accurately determine the antibody
level resulting from vaccination or
naturally infection with measles virus.
Several enzyme immune assay (EIA)
kits for anti-measles virus antibodies
are available commercially, among
which the Dade Bering EIA kit was
previously found to perform better in
comparison with other commercial
EIA kits [10]. Therefore this kit was
used in evaluation of the immune
response in this work.
The objectives of present study were:
1. To
evaluate
measles-specific
humeral immune response IgM in
teenager (Dentistry students), as well
as children naturally infected with
measles virus.
2. To differentiate between primary
and secondary immune failure by
detection
the
avidity
of
immunoglobulin G.
Materials and Methods
During March to June 2009
several cases of measles were detected
in
Babylongovernorate
including
teenager university student, and
childrens. The first case came from
Wasit governorate who live in student
resident house, in Hilla city. Most
students in contact with him became ill
and seafaring from fever, cough and
coryza most of them after 3 days later
developed a generalized maculopapuler rash and was subsequently
confirmed by serological study to have
measles. Information about students'
illness, vaccination status and their age
were reported. Blood samples by vein
puncture at acute and convalescent
period were collected to test for the
presence
of
measlesspecific
antibodies .
Serological analysis
Serum were separated from collected
blood samples , they were tested for
the presence of measles specific IgM
and IgG antibodies by using
previously described indirect enzyme
immunoassay that have been shown to
be highly sensitive (99.6%)and highly
specific
(100%){Enzygot,
Dade
Behring,
Marburg,
Germany}
(Hamkar R. et al., 2006).
Study group samples
Nine sera sample were
collected from laboratory confirmed
measles patients aged ≤ 2 years old
whom were received single dose of
measles virus ( except two of them of
unknown
vaccination
status)
experienced primary infection, and
were not re infected by measles virus,
therefore their sera were regarded as
low avidity anti- measles IgG.
Another 7 sera sample were
collected from subjects (3 male and 4
female) age 12 years old who had been
vaccinated twice for measles at 9
months with measles vaccine and 15
months of age with MMR vaccine
during vaccination program in Hilla
city Babylon governorate (one of them
of unknown vaccination status).
The third group of sera sample were
colleted from 36 university student
aging from 19 – 25 years whom were
suffering from acute measles infection
regarded as having high avidity IgG
against measles virus.
At the time of sera sample
collection, a question about personal
data,
vaccination
status
was
completed. Cases were confirmed as
measles when testing for anti-measles
IgM gave positive IgM results when
using Enzygost anti-measles IgM and
IgG (EIA) kits (Dade Behring,
Marburg, Germany) .
Avidity measurement
All
sera
samples
were
subjected to anti-measles IgG avidity
assay according to [2],[7];[8]. The
avidity of IgG for measles virus was
measured by a protein- denaturing
enzyme immune assay where the
antibodies were first allowed to bind to
the virus antigen followed by elution
with or without, six molar urea. Each
sample was tested at dilution of 1:21
and each sample at 4 replicates. For
each replicate a single serum dilution
(1:21) of the kit serum diluents was
applied to each of 4 wells on one row
of ELISA plates. (Two wells of
measles antigen positive - coated and 2
wells of measles antigen negative coated).
After incubation for 1 hour,
test plates were washed 4 times
according to ELISA kit procedure.
Then 2 wells (one antigen – positive
and one antigen – negative were
soaked for 5 minutes in wash buffer
containing 6 mol / L. urea. Fresh
buffer were applied and the soaking
was carried out twice more.
The plates was then washed 4
times with wash buffer. Then the test
was continued according to the kit
restriction manual. The remaining
specific antibody was then detected
according to the EIA kit procedure. An
avidity index (AI) was calculated from
the optical density (OD) of the wells
according to the following formula :
AI = (∆OD with urea / ∆OD with wash
buffer) ×100
Three control were used for
testing
EIA plates serum sample
containing strong high- avidity, weak
high avidity and low- avidity antimeasles IgG- antibody, in addition to
kit positive and negative control were
also applied.
Results
Serum samples included in this
study was grouped by age and measles
vaccination
status
and
their
distribution was shown in table (1).
All samples were tested and
confirmed as measles case in
laboratory,
(anti-measlesIgM
positive). Nine ≤ 2 year old children
and seven (12) years old subjects
samples were used as a control source
of low and high avidity from all age
groups, 10 (19.2%) had unknown
vaccination status, 8 (15.4%) had not
been vaccination, and 34(65.4%) had
received one (21 subjects) or two
(13subjects) doses of measles vaccine.
Overall, 24 (46.2%) measles
cases confirmed by a positive IgM test
exhibit high-avidity IgG representing
secondary immune response to
measles (that representing secondary
vaccine failure),while remaining other
28 subjects (53.8%)of them exhibiting
a primary immune response (low
avidity) indicating primary measles
infection see table (2). The table
reflected also anti-measles virus low
and high- avidity IgG which was
distributed as following: subjects less
than 2 years old show 100% low
avidity and this proportion decreased
gradually with increasing age and
reach 28.5% of those age 23 years.
High avidity anti - measles
virus IgG in higher proportion at
subjects age 23 years (71%) followed
by 22 years (70%) then 21 years(60%).
Whereas 2 cases (28.6%) was found in
12 years age group and non in ≤ 2
years age group .
The distribution of anti-measles virus
high and low-avidity IgG among the
study group by vaccination status was
reflected in table (3) below. Non of the
8 unvaccinated patients (0 %) reflect
high- avidity anti-measles IgG so their
infection was regarded as a primary
measles infection. Of the remaining 35
patients which were sourly vaccinated
15 (42.9%) showed low avidity ( i.e., a
primary immune response) and 20
(57.1%) showed a secondary immune
response, which indirectly reflecting a
secondary vaccine failure .
There was no relationship
between vaccination status and age(P
> 0.05), but there was a clear
relationship between anti-measles
virus IgG avidity with age (P < 0.05).
Also there was no relationship
between vaccination and avidity (P >
0.05) but there was a direct
relationship between number of
vaccine dose and avidity. Table (3)
also reflected that subjects who
received two dose of measles virus (
infected and vaccinated) showed highavidity anti-measles virus IgG
proportion (55%) , where as subjects
who received single dose vaccine
showed lower proportion (45%) .
Discussion
Although vaccination program
with measles virus live attenuated
virus vaccine in Iraq started since
1980s, by a routine immunization
program, under which the vaccine is
given in one dose scheduled at 9
months of age, with a recommendation
for vaccination at 18 month age with
MMR trivalent vaccine. yet measles
cases were reported every year
anywhere in Iraq and neighboring
countries, however during March 2009
a large episodes of measles infection
was seen and teenager subject were
included most of them had been
vaccinated, so this study was directed
mainly toward teenager patients
(dentistry student). Several research
published that vaccine- induce
protection with less duration less
robust
than
naturally acquired
immunity against measles virus
[7],[15], also high occurrence of mild
symptoms measles due to secondary
vaccine failure [4], [7]; [9]; [11]; [17];
[18] has been found among measles
patients vaccinated over decade age
especially among those who were
revaccinated [7],[17]. Hence the
evolution
of
measles
control
programmes in Iraq and neighboring
countries require well understand of
the resions for primary and secondary
vaccine failure. The presence of high
proportion of primary vaccine failures
in vaccinated patients with measles,
give an indication of improper vaccine
handling, for example, improper cold
chain or misvaccination, in addition to
suppressing factors influencing Iraqi
population since more them 3 decades,
besides
persistent
exposure
to
naturally circulating measles virus. All
this gave reasons to follow up the
immune status of Iraqi population, to
improve measles control in Iraq.
Therefore introduction of good
diagnostic test for detection of measles
such as IgM- capture EIA for detection
of measles IgM is not sufficient to
differentiate between primary infection
and re infection due to second vaccine
failure [7],[17]. Detection of specific
measles IgM detected only in primary
measles infection or vaccination but
also inducible by overcame end.
Few researcher used IgG
avidity test as a useful procedure for
identifying primary and secondary
immune responses [7], yet few reports
upon this test used during measles
outbreaks [7],[17]. However in this
study 46.2 % of 52 of measles cases
confirmed by a positive IgM EIA test
mounted a secondary immune
response, giving an indication that
presence of IgM can not be used as a
reliable indicator of a primary immune
response [7],[17]. The present study
also reflected that all unvaccinated
subjects showed a primary immune
response supporting the information
giving by the IgG avidity test. This
finding inconsistent with other
researchers, and I had concluded that:
1. measles virus can infect previously
immune responded persons, producing
classic symptoms of measles in some,
and mild or no symptoms in others.
2. The protective immunity induced by
vaccination may not be life long
without being boosted by an exposure,
mostly sub clinically, to a naturally
circulating virus.
3. Due partly if not entirely, to the
secondary vaccine failure, the numbers
of measles cases among adults in Iraq
increase in recent over the previous
year.
4. Measles is still endemic in Iraq, and
person has the potential for repeated
exposure to wild type measles virus.
1- Large scale serological estimation
of immune status of Iraqi population is
required in all governorates to find the
population requiring vaccination to be
included in rotten vaccination program
2-Implementation of better vaccination
programs
is
urgently
required
especially for those aging 15-20years
as a boaster dose.
3-IgG immunoglobulin avidity test is a
good parameter for detection of
primary and secondary immune
response failure.
Table1 Distribution of anti-measles IgM-positive cases by age and measles
vaccination status
Age
(years)
≤2
12
19
20
21
22
23
Total
Total
subject
Tested
9
7
6
8
5
10
7
52
vaccination status
unknown
Known
No.
%
No
%
2
1
2
1
0
2
2
10
22.2
14.3
33.3
12.5
0
20
28.6
19.2
7
6
4
7
5
8
5
42
77.8
85.7
66.7
87.5
100
80
71.4
80.8
No. of vaccine
doses(Known
vaccination status)
0
No.
2
0
1
2
0
2
1
8
1
No.
2
0
2
4
4
5
4
21
2
No.
3
6
1
1
1
1
0
13
Table 2 Distribution of low and high- avidity anti-measles virus IgG in the study group by
age.
anti-measles virus IgG
Age
(years)
Total
subject
Tested
Low- avidity
high- avidity
No.
No.
%
%
≤2
9
9
100
0
12
7
5
71.4
2
19
6
3
50
3
20
8
4
50
4
21
5
2
40
3
22
10
3
30
7
23
7
2
28.5
5
Total
52
28
53.8
24
Table 3 Distribution of low and high- avidity anti-measles virus
group by vaccination status.
antimeasles
virus
IgG
Total
subject
Tested
No.
0
28.6
50
50
60
70
71
46.2
IgG in the study
known vaccination status
(No. of doses of vaccine)
vaccination status
unknown
known
No.
%
No
%
0
1
2
No.
%
No. %
No. %
Highavidity
24
4
44.4
20
46.5
0
0
9
60
11
55
Lowavidity
28
5
55.6
23
53.5
8
100.0
6
40
9
45
Total
52
9
100.0
43
100.0
8
100.0
15
100.0
20
100.0
References
1- Bin D., Zhihui C.; Qichang L.;et al.1991. Duration of immunity following
immunization with live measles vaccine: 15 yearsof observation in Zhejiang
Province, China. Bull WHO.69:415-23.
2- Chen, R. T., L. E. Markowitz, P. Albrecht, J. A. Stewart, L. M. Mofenson, S. R.
Preblud, and W. A. Orenstein. 1990. Measles antibody: reevaluation of protective
titers. J. Infect. Dis. 1621036-1042.
3- De Souza V. U.A, Pannuti C.S.; Masami S.; and De Andrade H.F. 1997. Enzymelinked immunosorbent assay-IgG antibody avidity test for single sample serologic
evaluation of measles vaccines. Journal of medical virology. 52:275–9.
4- Erdman, D. D., J. L. Heath, J. C. Watson, L. E. Markowitz, and W. J. Bellini.
1993. Immunoglobulin M antibody response to measles virus following primary and
secondary vaccination and natural virus infection. J. Med. Virol. 1:44-48.
5- Griffin, D. 2001. Measles virus, p. 1401-1441. In D. M. Knipe and P. M. Howley
(ed.), Fields virology, 4th ed. Lippincott Williams and Wilkins, Philadelphia, Pa.
6- Hamkar, R., M. Mahmoodi, R. Nategh, K.N. Jelyani, M.B. Eslami and T. Mohktari
-Azad. 2006 Distinguishing between primary measles infection and vaccine failure
reinfection by IgG avidity assay; Health journal. 12 (6):775-782.
7- -Hamkar, R., M. Mahmoodi, R. Nategh, K.N. Jelyani, M.B. Eslami and T.
Mohktari-Azad. 2006. Distinguishing between primary measles infection and vaccine
failure reinfection by IgG avidity assay; Health journal . 12 (6):775-782.
8- Hedman, K., and S. A. Rousseau. 1989. Measurement of avidity of specific IgG for
verification of recent primary rubella. J. Med. Virol. 27:288-292.
9- Helfand, R. F., J. L. Heath, L. J. Anderson, E. F. Maes, D. Guris, and W. J. Bellini.
1997. Diagnosis of measles with an IgM captures EIA: the optimal timing of
specimen collection after rash onset. J. Infect. Dis. 175:195-199.
10- Hesketh, L., A. Charlett, P. Farrington, E. Miller, T. Forsey, and P. Morgan
Capner. 1997. An evaluation of nine commercial EIA kits for the detection of measles
specific IgG. J. Virol. Methods 6651-59.
11- Hidaka, Y., T. Aoki, M. Akeda, C. Miyazaki, and K. Ueda. 1994. Serological and
clinical characteristics of measles vaccine failure in Japan. Scand. J. Infect. Dis.
26:725-730.
13- Krugman S1983. Further-attenuated measles vaccine: characteristic and use.
Rev. Infect.Dis.5:477-81
14- Moss, W. J., and D. E. Griffin. 2006. Global measles elimination. Nat. Rev.
Microbiol. 4900-908.
15- Mossong J, Muller CP. 2003. Modelling measles re-emergence as a result of
waning of immunity in vaccinated populations. Vaccine. 21:4597–603.
16- Narita, M., S. Yamada, Y. Matsuzono, O. Itakura, T. Togashi, and H.
Kikuta.1996 Immunoglobulin G avidity testing in serum and cerebrospinal fluid for
analysis of measles virus infection. Clinical and diagnostic laboratory immunology.
3:211–5.
17- Pannuti, C. S., R. J. Morello, J. C. de Moraes, S. P. Curti, A. M. S. Afonso, M. C.
C. Camargo, and V. A. U. F. de Souza. 2004. Identification of primary and secondary
measles vaccine failures by measurement of immunoglobulin G avidity in measles
cases during the 1997 São Paulo epidemic. Clin. Diagn. Lab. Immunol. 11:119122.44
18- Paunio, M., K. Hedman, I. Davidkin, M. Valle, O. P. Heinonen, P. Leinikki, A.
Salmi, and H. Peltola. 2000. Secondary measles vaccine failures identified by
measurement of IgG avidity: high occurrence among teenagers vaccinated at a young
age. Epidemiol. Infect. 124:263-271.
19- Poland, G. A., and R. M. Jacobson. 1994. Failure to reach the goal of measles
elimination. Apparent paradox of measles infections in immunized persons. Arch.
Intern. Med. 1541815-1820.
20- Ratnam, S., V. Gadag, R. West, J. Burris, E. Oates, F. Stead, and N.Boullianne.
1995. Comparison of commercial enzyme immunoassay kits with plaque reduction
neutralization test for detection of measles virus antibody. J. Clin. Microbiol. 33:811815
21- Tuokko H. 1995.Detection of acute measles infections by indirect and mu-capture
enzyme immunoassays for immunoglobulin M antibodies and measles
immunoglobulin G antibody avidity enzyme immunoassay. Journal of medical
virology. 45:306–11.